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. 2015 Feb 20;43(9):e57. doi: 10.1093/nar/gkv124

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Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Figure 6. — (a) A scheme of the TAR-BRV- tTA^VP64 vector. This vector contains restriction sites for insertion of two unique targeting sequences (hooks) (dot red) homologous to 5' and 3' ends of a gene of interest and restriction sites for insertion of chromatin insulators to protect a gene from silencing (dot green). The vector also contains a YAC cassette that includes a yeast selectable marker HIS3 for selection in yeast and a yeast centromere CEN6 for proper segregation in yeast cells. The TAR-BRV- tTA^VP64 vector also contains a BAC cassette that includes a F’ factor origin of replication and the chloramphenicol acetyltransferase (CmR) gene for selection in bacterial cells. For gene TAR cloning experiments, the TAR-BRV- tTA^VP64 vector DNA should be linearized by a unique endonuclease located between the hooks to expose targeting sequences (hooks). The TAR-BRV- tTA^VP64 vector also contains a 3' HPRT-loxP cassette allowing a further loading of a TAR-gene isolate into the unique loxP site of the alphoid^tetO-HAC in hamster CHO cells by Cre/loxP mediated recombination, from where the HAC may be MMCT-transferred to the recipient human cells for further gene function analysis. The TAR-BRV- tTA^VP64 vector also contains the tTA^VP64 chromatin modifier that is constitutively expressed from the HAC. In the presence of doxycycline the tTA^VP64 cannot bind to the tetO-sequences in the alphoid^tetO-HAC. (b) FISH analysis of the HAC containing the TAR-BRV- tTA^VP64 vector in HT1080 cells. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a vector probe (red).